Selective response of human airway epithelia to luminal but not serosal solution hypertonicity. Possible role for proximal airway epithelia as an osmolality transducer.

The response of cultured human nasal epithelia to hypertonic bathing solutions was tested using ion-selective microelectrode and quantitative microscopy. Raised luminal, but not serosal, osmolality (+/- 150 mM mannitol) decreased Na+ absorption but did not induce Cl- secretion. Raised luminal osmolality increased cell Cl- activity, Na+ activity, and transepithelial resistance and decreased both apical and basolateral membrane potentials and the fractional resistance of the apical membrane; equivalent circuit analysis revealed increases in apical, basolateral, and shunt resistances.

Excitotoxic lateral pallidotomy does not relieve L-dopa-induced dyskinesia in MPTP parkinsonian monkeys.

We attempted to relieve the marked overactivity known to occur in the lateral segment of the globus pallidus (GPL) in L-DOPA-induced dyskinesia (LID) by unilateral stereotaxic ibotenic acid lesioning of the GPL in 4 monkeys with MPTP-induced parkinsonism. Two already dyskinetic animals were pallidotomized to counteract LID once established, while 2 L-DOPA-naive MPTP-treated animals were pallidotomized before L-DOPA was ever administered in an attempt to prevent the development of the process conductive to LID. Acutely after the lesion, more prominent akinesia (particularly in the contralateral limbs) with contraversive body deviation and circling behavior were seen for 48 h.

Anomalies in ion transport in CF mouse tracheal epithelium.

The cystic fibrosis (CF) mouse trachea has become a model for gene transfer. To characterize ion transport properties of tracheal epithelium from normal and CF mice, tracheas were excised, mounted in Ussing chambers, and basal properties and responses to pharmacological agents and/or ion substitution protocols measured. No difference in basal short-circuit (Isc) was observed between normal (29.

Interaction of bioactive hydrophobic peptides with the human multidrug transporter.

In this report we demonstrate that various biologically active hydrophobic peptide derivatives, e.g., proteinase inhibitors, chemoattractants, ionophores, enkephalins, and immunosuppressants, stimulate a membrane ATPase activity associated with the human multidrug transporter (MDR1).

Tracheal vascular response to hypertonic and hypotonic solutions.

One of the major roles of the upper respiratory mucosa is to humidify inspired air. This function requires the coordinated activity of respiratory epithelium and mucosal vasculature. It has been difficult to study this relationship in vivo.

Gene therapy for cystic fibrosis using E1-deleted adenovirus: a phase I trial in the nasal cavity. The University of North Carolina at Chapel Hill.

Cystic fibrosis (CF) is an autosomal recessive disease that reflects mutations in the CFTR gene. Multiple mutations in this gene have been detected that lead to a protein (CFTR) that is abnormally metabolized, dysfunction, or both. The full spectrum of the activities of the gene product have not been defined, but it is clear that CFTR can act as a cAMP-regulated Cl- channel.

Hyperabsorption of Na+ and raised Ca(2+)-mediated Cl- secretion in nasal epithelia of CF mice.

We investigated the effect of homozygous genetic disruption of the murine cystic fibrosis transmembrane regulator (CFTR) gene on regulation of the rates of Na+ absorption and Cl- secretion by nasal epithelia in cystic fibrosis (CF) mice. The basal in vivo nasal potential difference (PD; -28.8 +/- 1.

Cloning and expression of a human P2U nucleotide receptor, a target for cystic fibrosis pharmacotherapy.

The Cl- secretory pathway that is defective in cystic fibrosis (CF) can be bypassed by an alternative pathway for Cl- transport that is activated by extracellular nucleotides. Accordingly, the P2 receptor that mediates this effect is a therapeutic target for improving Cl- secretion in CF patients. In this paper, we report the sequence and functional expression of a cDNA cloned from human airway epithelial (CF/T43) cells that encodes a protein with properties of a P2U nucleotide receptor.

Mechanism of sodium hyperabsorption in cultured cystic fibrosis nasal epithelium: a patch-clamp study.

Transepithelial Na+ absorption is increased two to three times in cystic fibrosis (CF) compared with normal (NL) airway epithelia. This increase has been associated with a higher Na+ permeability of the apical membrane of airway epithelial cells. Because Na+ absorption is electrogenic and abolished by amiloride, Na+ channels are thought to dominate the apical membrane Na+ permeability.

Pyridine nucleotide redox potential modulates cystic fibrosis transmembrane conductance regulator Cl- conductance.

Cl- conductance of the apical membrane of airway epithelial cells has properties of a passive diffusion mechanism but is decreased by inhibition of oxidative metabolism. Recent reports that cAMP-dependent Cl- conductance also requires ATP at the intracellular domains of the cystic fibrosis transmembrane conductance regulator (CFTR) suggests that ATP concentration could mediate metabolic regulation of Cl- conductance. However, metabolic inhibitors affect processes other than ATP free energy levels, including notably the metabolic pathways that set the redox potential of pyridine nucleotides within the cell.

Calcium-dependent release of arachidonic acid in response to purinergic receptor activation in airway epithelium.

The effect of purinergic receptor agonists on arachidonic acid release was investigated in [3H]arachidonic acid-prelabeled human airway epithelial cells. Exposure of bronchial epithelial BEAS39 cells to extracellular ATP resulted in a marked release of unesterified [3H]arachidonic acid with maximal effect observed within 60-90 s. [3H]diacylglycerol and [3H]phosphatidic acid accumulated in parallel with [3H]arachidonic acid.

Relationship of a non-cystic fibrosis transmembrane conductance regulator-mediated chloride conductance to organ-level disease in Cftr(-/-) mice.

Although loss of cystic fibrosis transmembrane conductance regulator (CFTR)-mediated Cl- channel function is common to all epithelia in cystic fibrosis (CF) patients, the severity of disease varies in different organs. We hypothesized that differences in disease severity in CF relate to the expression of an "alternative" plasma membrane Cl- conductance. In CF mice [Cftr(-/-); mice homozygous for Ser-489 to Xaa mutation], which do not express cAMP CFTR-mediated Cl- secretion, we surveyed organs that exhibit a range of disease severity for a Ca(2+)-mediated apical membrane epithelial Cl- conductance.

[Prospects of gene therapy in mucoviscidosis using viral infection of the airway epithelium].

Mucoviscidosis is the most common severe inherited autosomal recessive disease. Since the gene has been recognised (cystic fibrosis transmembrane conductance regulator gene) (CFTR) the technique of genetic transfer has been applied to the airway epithelium. The prospect for gene therapy to treat the consequences of bronchopulmonary mucoviscidosis is now evident.

Synthesis and vectorial export of cGMP in airway epithelium: expression of soluble and CNP-specific guanylate cyclases.

Guanosine 5'-cyclic monophosphate (cGMP) is an important modulator of fluid balance in many epithelia. We examined its metabolism in primary cultures of human airway epithelia. Sodium nitroprusside increased cGMP levels 30-fold, suggesting that the respiratory epithelium expresses a soluble guanylate cyclase; however, endogenous nitric oxide production was not detected.

Control of the mucosal microcirculation in the upper respiratory tract.

This study was designed to investigate the regulatory mechanisms of the mucosal microvascular network in the upper respiratory tract. Tracheal mucosal circulation was observed using a specially constructed chamber that allowed direct microscopic visualization of mucosal arterioles. Solutions of increasing hypertonicity (500 and 900 mOsm) applied to the tracheal epithelium resulted in increasing dilation of the underlying mucosal arterioles (p < 0.

Amiloride inhalation therapy in cystic fibrosis. Influence on ion content, hydration, and rheology of sputum.

Amiloride inhalation as treatment for cystic fibrosis (CF) lung disease has been shown in independent studies to increase mucus clearance by ciliary and/or cough action and to retard the decline in lung function. It is hypothesized that amiloride therapy decreases the excess sodium and water absorption that is a characteristic of CF airway epithelium and that it leads to an improvement in the rheologic properties of mucus favoring airway mucus clearance. The aim of this study was to investigate whether amiloride treatment (5 x 10(-3) M amiloride in one-third normal saline four times a day) would change sputum electrolyte composition in patients with CF after 25 wk of therapy as compared with placebo (one-third normal saline), and whether appropriate changes in sputum water content and rheologic properties would accompany any changes in electrolyte composition.

Sodium-permeable channels in the apical membrane of human nasal epithelial cells.

We used patch-clamp techniques to study the channels that underlie the Na+ conductance of the apical membrane of human normal nasal epithelial cells. Cells were cultured on permeable supports and studied after confluence. In 172 of 334 (52%) excised membrane patches, we observed 20-pS Na(+)-permeable channels that do not discriminate between Na+ and K+ (pNa/pK = 1.

Functional consequences of heterologous expression of the cystic fibrosis transmembrane conductance regulator in fibroblasts.

We studied the consequences of cystic fibrosis transmembrane conductance regulator (CFTR) expression in NIH-3T3 fibroblasts as a model for the effects of virally transduced CFTR expression in non-epithelial cells. Fibroblasts were infected with a retrovirus vector that contained the human CFTR and neor cDNAs. We selected and expanded G418-resistant clones that encompassed a range of CFTR expression.

Nucleotide regulation of goblet cells in human airway epithelial explants: normal exocytosis in cystic fibrosis.

The regulation of mucin secretion by airway goblet cells is poorly understood and the receptor-based regulatory mechanisms have not been described in human airways. In the present study, we report that extracellular triphosphate nucleotides regulate the rate of granule release from goblet cells in both normal and cystic fibrosis (CF) airway epithelial explants. Explants isolated from nasal and tracheobronchial tissues were mounted in perfusion chambers and the secretory activity was assessed by videomicroscopic determination of degranulation in single goblet cells and by ELISA determination of mucins secreted into the mucosal perfusate.

Enhanced Na+ transport in an air-liquid interface culture system.

Use of the air-liquid interface culture technique has produced improved morphological differentiation of rodent, canine, and human tracheal epithelia. We have investigated the effect of this culture technique on ion transport activities of cultured canine bronchial epithelia. These cells were isolated from excised airways by enzymatic digestion and plated on permeable collagen membrane substrates.

CFTR and outward rectifying chloride channels are distinct proteins with a regulatory relationship.

In cystic fibrosis (CF), numerous epithelial cell functions are abnormal, including Cl- conductance, sodium absorption, mucin sulphation and enzyme secretion. Although the CF gene product, the cystic fibrosis transmembrane conductance regulator (CFTR), functions as a small linear Cl- channel, it is difficult to attribute such pleiotropic disease manifestations solely to a defect in Cl- conductance. This has led to speculation that CFTR regulates the activity of other proteins.

Papilloma virus immortalized tracheal epithelial cells retain a well-differentiated phenotype.

Human airway epithelial cell lines that retain phenotypic properties representative of the native tissue will be useful physiological models. Human papilloma viral (HPV) genes can immortalize human genital keratinocytes and breast and bronchial epithelia. We transfected cystic fibrosis (CF) and normal tracheobronchial epithelial cell cultures with DNA encoding the HPV-18 E6 and E7 genes and characterized phenotypic properties of resultant cell lines.

Sulfate concentrations and transport in human bronchial epithelial cells.

Inorganic sulfate concentrations in the cytoplasm of human bronchial epithelial cells exceeded levels in the bathing medium under all circumstances tested. Cell sulfate concentrations were directly related to medium sulfate concentrations and inversely related to medium chloride concentrations. In physiological media there was a sulfate compartment of approximately 0.